Alterations to chromatin dynamics and genome organization have important consequences on organismal development and gene expression. Mutations to chromatin machineries that are responsible for transmission of epigenetic signals and for the deposition and removal of histones (i.e., histone modifying enzymes and chaperones) cause genomic changes and chromosomal rearrangements that are frequently observed in cancer. Histone variants are associated with specific chromatin states, and in particular, the histone H3 variant CENP-A (Cse4 in yeast) is vital for centromere formation. To prevent the formation of multicentric chromosomes and aneuploidy, ectopically deposited CENP-A is targeted for proteosomal degradation by the E3 ligase Psh1. We have recently discovered a connection between the histone chaperone Spt6 and Psh1. Spt6, a positive regulator of transcriptional elongation, is important for maintaining histone modification profiles in the wake of transcription. The mechanism by which Psh1 identifies ectopic Cse4 is not well understood, and the role of Spt6 in this process is unexplored. My hypothesis is that Spt6 plays a vital role in maintaining centromere identity through association with Psh1, thus aiding in the removal of aberrantly deposited Cse4 that would otherwise result in massive genome instability. I further hypothesize that the replacement of centromeric Cse4 in S-phase is mediated by Spt6-dependent transcription through the centromere. To test these hypotheses, I will use the model organism Saccharomyces cerevisiae to: (1) Define the interacting domains and importance of the Spt6:Psh1 complex in genome stability. (2) Determine the role of Spt6 in the prevention of ectopic Cse4 deposition within transcription units. (3) Explore the link between cell cycle-dependent disruption of centromere chromatin and Spt6. Using a combination of biochemical approaches and genome integrity assays, I will confirm the Spt6:Psh1 interaction in budding yeast in vitro and in vivo. Chromatin immunoprecipitation studies (genome wide and single locus) will be used to determine the transcription-dependent contribution of Spt6 to the removal of ectopic Cse4. Lastly, NET-seq (nascent elongating transcript-deep sequencing) and fluorescent pulse-chase experiments will explore the role of Spt6-mediated transcription on S-phase replacement of centromeric Cse4. The research outlined in this proposal will advance our understanding about the mechanisms that contribute to proper centromere maintenance and stability. Importantly, increased genome instability and aneuploidy that result from centromere disruption are hallmarks of cancer. Completion of these studies in yeast will provide a firm understanding of the mechanism of these processes, which can be expanded to studies in human cancer models.